Beta-adrenergic receptors (BARs) belong to the G protein-coupled receptor (GPCR) superfamily and activate adenylyl cyclase via the stimulatory G protein (Gs) in response to the endogenous catecholamines epinephrine and norepinephrine. We have been investigating the regulation of this signaling pathway and focused on differences in regulation of the three beta-subtypes, B1AR, B2AR and B3AR. B2AR is more susceptible to regulation than is B1AR whereas B3AR is very resistant. This may provide a physiological basis for the existence of three subtypes. To compare the regulation of B1AR and B2AR, we used baby hamster kidney (BHK) and human embryo kidney (HEK 293) cells expressing similar levels of either subtype. Previously, we showed that: B1AR undergoes less agonist-stimulated phosphorylation, desensitization, internalization and down-regulation than B2AR. Phosphorylation of the C-tail of B2AR by GPCR kinases (GRKs) is required for arrestin binding to the receptor resulting in desensitization. In addition, arrestins bind clathrin and promotes receptor internalization. B1AR also is endocytosed by the clathrin-coated pit pathway but the two subtypes are sorted to different endosomal compartments. Upon removal of agonist, both subtypes recycle at similar rates back to the plasma membrane. Upon prolonged agonist treatment, B2AR undergoes more down-regulation than B1AR. Using chimeras in which the C-tails have been exchanged, we demonstrated that the C-tail is the major determinant of subtype differences in these regulatory processes. It has been shown for several GPCRs that agonist stimulation leads to ubiquitination of the receptor. As ubiquitin serves as a signal for sorting the receptors to lysosomes, we determined whether B1AR is ubiquitinated. We readily observed the ubiquitination of B2AR that was both time- and agonist-dependent, and enhanced in cells treated with proteasomal inhibitors. In contrast, ubiquitination of B1AR was not detectable even under these optimal conditions. Moreover, in cells expressing BAR chimeras in which the C-termini have been switched, the chimera of B1AR with the B2AR C-tail underwent ubiquitination and down-regulation but that of B2AR with the B1AR C-tail did not. Our results demonstrate for the first time that B1AR and B2AR differ in the ability to be ubiquitinated. The lack of agonist-mediated ubiquitination of B1AR may prevent extensive trafficking of the receptor to lysosomes and thus account for its resistance to down-regulation. As ubiquitination of other receptors is linked to their phosphorylation, our results are consistent with our basic hypothesis that the limited GRK-catalyzed phosphorylation of B1AR is the key event in determining its regulatory fate. We also are pursuing the ubiquitination of B2AR by identifying the lysine residues that are ubiquitinated. The B2AR has 14 lysine residues in its intracellular domains: 1, 3 and 7 in intracellular loops 1, 2 and 3; and 3 in the C-tail. Although the C-tail is the most likely region for ubiquitination, a lysine in the distal part of the 3rd loop of the vasopressin V2 receptor is the only ubiquitination site for that GPCR. We are testing two mutant B2ARs, K270R (3rd loop) and K348R, K372R, K375R (C-tail). K270R behaved as the wild-type B2AR when assayed for agonist-mediated internalization and down-regulation whereas the triple mutant exhibited increased internalization and reduced down-regulation. These results suggest that the C-tail of B2AR is ubiquitinated.